DETERMINING CANINE proBNP

ABSTRACT

Methods and compositions are disclosed for determining canine proBNP or fragments thereof in a sample. In one method, canine proBNP or fragments thereof are determined by providing a canine sample, contacting the sample with at least one antibody that binds an epitope in the region from amino acids 1 to 22 of canine proBNP, and determining the presence of the canine proBNP or fragments thereof present in the sample. Antibodies that bind canine proBNP and kits comprising such antibodies are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/596,968 filed on 30 Jun. 2006, which is a U.S. national phaseapplication under 35 U.S.C. § 371 of International Application No.PCT/EP2005/054446 filed 8 Sep. 2005, which claims priority to AustrianApplication No. A 1505/2004 filed 8 Sep. 2004. The entire text of eachof the above-referenced disclosures is specifically incorporated byreference herein without disclaimer.

BACKGROUND

The present invention relates to a method of determining proBNP orfragments thereof in mammals.

Heart diseases play an important part not only in humans, but alsoanimals, in particular pets, such as dogs or cats, are afflicted withthese diseases. Studies have shown that, e.g., each tenth canine hearthas a functional impairment. The heart diseases occurring concern, forinstance, the cardiac valves and the cardiac muscle. Since at first theheart is capable of compensating functional impairment by workingharder, such a disease in most cases remains hidden, with theconsequence that the state of the heart will deteriorate due to theincreased load on the heart. The symptoms resulting from heart diseases,such as fatigue, circulatory insufficiency, languor, can mostly berecognized when the pet's heart is no longer able to compensate theweakness. In such a case, the heart disease has already progressed somuch that complete curing is hardly possible any more.

As a rule, chronic cardiac valve and cardiac muscle changes are notcurable, yet by the use of medicaments, the further progress of theheart disease can be slowed down. Therefore, an early diagnosis must bemade for the occurring heart diseases. By way of routine, mainlyphysical methods are used for this purpose, such as auscultation of theheart sounds, the recording of an electrocardiogram, X-ray andultrasonic examinations. These examination methods mainly have thedisadvantage that they can be carried out only when already visible oraudible defects can be directly recognized on the heart. Furthermore,physical examination methods require suitable and, as a rule, expensivedevices in order to carry out a respective diagnosis.

The heart diseases most frequently occurring in dogs, e.g., are heartdecompensation and dilated cardiomyopathy, which mainly afflict biganimals. Dilated cardiomyopathy is a heart disease which causes anenlargement of the ventricles of the heart with normal wall thickness,such enlargement quickly causing cardiac insufficiency in the afflictedanimal. By admixing taurine to the feed, the risk of falling ill withdilated cardiomyopathy could be reduced significantly. In an illnessrelated to dilated cardiomyopathy, the restrictive cardiomyopathy whichfrequently is found in older cats, a continuous decrease in the heartfunction with a reduced ability for pumping can be observed. The heartdisease most frequently occurring in cats is hypertrophiccardiomyopathy. This disease of the cardiac muscle causes thickening ofthe heart wall and a resultant reduced ability to fill the ventricles ofthe heart with blood. This leads to an accumulation of blood in the leftventricle and to a greatly reduced amount of blood being pumped throughthe body.

In many heart diseases, such as, e.g., cardiac insufficiency, dilatedcardiomyopathy, hypertrophic cardiomyopathy, left-ventricularhypertrophy and dysfunction, a peptide hormone, the so-called BNP (brainnatriuretic peptide) is secreted. This hormone causes the excretion ofliquid via the kidneys and thus regulates the cardiovascular system.Since this peptide is produced in the heart and is increasingly producedin case of an overstress and congestion of the heart, determining theBNP level in blood is a suitable means for evaluating cardiacinsufficiency.

BNP as well as other natriuretic peptides play an important part inregulating the water balance and the blood pressure. If the cardiac wallis dilated, it secrets BNP in increasing amounts, causing an excretionof sodium and liquid via the kidneys and a dilation of the bloodvessels, which in sum can lower the blood pressure and the filling levelof the heart. BNP is synthesized by the cells of the cardiac muscle asproBNP which finally is cleaved into n-terminal proBNP and BNP. Bothparts of the BNP are delivered to the blood and can be determinedtherein.

Cardiac diseases in animals are, inter alia, dealt with in the followingpertinent publications: Bright J M and Cali J V, J Am Vet Med Assoc2000, 216:1110-4; Guglielmini C, Vet Res Commun 2003, 27 Suppl 1:555;Boswood A et al., J Small Anim Pract 2003, 44:104-8; Takemura N et al.,J Vet Med Sci 2003, 65:1265-7; MacDonald K A et al., J Vet Intern Med2003, 17:172-7; Greco D S et al., Can Vet J 2003, 44:293-7; Monnet E etal., J Am Vet Med Assoc 1997, 211:569-72; Hamlin R L et al., J VetIntern Med 1996, 10:85-7; Gaschen L et al., J Vet Intern Med 1999,13:346-56.

A large number of methods are already known in the prior art whichassist in the detection of human proBNP or the fragments thereof,respectively, in the serum of an individual. By way of example, here EP0 648 228 B1, WO 03/87819 and FR 2 843 396 should be mentioned.

In US 2004/0018577, an immunoassay is disclosed which comprises at leastthree antibodies which all are capable of binding to different epitopesof an analyte. The analytes to be detected particularly concern thedetection of markers regarding heart diseases, wherein i.a. also BNP andproBNP can be detected.

Biondo A. W. et al. (Vet. Pathol. 2003, 40(5):501-506) describe a methodof detecting ANP and BNP in cats by means of polyclonal antibodies whichare directed against a peptide of the ANP which comprises the aminoacids 1 to 28, and against a peptide which comprises the amino acids 43to 56 of proBNP, respectively.

In EP 1 016 867 A1, an immunoassay is described for the detection ofpreproBNP in mammals. There, antibodies are used which are directedagainst peptides comprising the amino acids 27 to 102, 73 to 102 and 27to 64 of human BNP.

Jortani S. A. et al. (Clin. Chem. 2003, 50(2):265-278) describe the useof BNP and its prepro- and pro-forms as possible markers for heartdiseases. In this article, no preferred peptide regions of BNP arementioned which would be suitable for detecting heart diseases in dogsand cats.

In WO 2000/35951 several peptides are disclosed against which antibodiescan be prepared, which are suitable in a method for diagnosing heartdiseases. Three peptides comprising the amino acids 1 to 13, 37 to 49,and 65 to 76 of human Nt-pro-BNP protein are disclosed there, which mayalso be used for preparing antibodies that are directed against thesepeptides.

Moreover, several test kits for detecting human proBNP or the fragmentsthereof, respectively, are commercially available (e.g. from Roche andBiomedica). Nevertheless, there is no known method with whose assistancespecifically proBNP in animal samples can be determined. Therefore, andbecause of the costly and complicated physical examinations of animalsit is an object of the present invention to provide suitable means fordetermining proBNP or the fragments thereof, respectively.

SUMMARY OF THE INVENTION

Therefore, the present invention provides a method of determining felineor canine proBNP or fragments thereof, comprising the steps of

providing a feline or canine sample,

contacting the sample with at least one antibody which, when determiningfeline proBNP, or fragments thereof, binds to at least one epitope inthe region comprising the amino acids 20 to 42 and/or in the regioncomprising the amino acids 57 to 80 of feline proBNP, and whendetermining canine proBNP, or fragments thereof, binds to at least oneepitope in the region comprising the amino acids 20 to 86 of canineproBNP, and

determining the presence and/or concentration of the feline or canineproBNP, or fragments thereof, present in the sample.

It has been found that an antibody which can bind to an epitope in thedisclosed regions of the feline, or canine, proBNP, respectively, isvery well suited to specifically determine proBNP.

It is pointed out that the feline and canine proBNP sequences hereindisclosed have been used by way of example for the family of thefelidae, or canidae, respectively, and that therefore individual aminoacids which differ from the sequences disclosed herein, in the proBNPsequences of animals of other species of these families also fall withinthe scope of the sequences disclosed herein as long as these differingamino acids do not relate to the epitopes of the anti-bodies disclosedherein in a manner that a specific binding is no longer renderedpossible. The amino acid sequences disclosed herein have been publishedin public data bases (e.g. Swiss-Prot: canine BNP-P16859 and felineBNP-Q9GLK4).

The samples used in the method according to the invention comprise fluidsamples, such as, e.g., blood, urine, as well as tissue samples, suchas, e.g., tissue sections of the cardiac muscle or of the brain. Asrequired, the samples may be processed accordingly, so as to facilitateor render possible e.g. the later contacting of the sample with theantibodies according to the invention. Thus, fractions containing proBNPor fragments thereof, respectively, can be provided from blood samples,or also tissue samples may, e.g., be homogenized and likewise beseparated from non-proteinaceous fractions.

The binding of at least one antibody to an epitope of the feline orcanine proBNP in the sample means that the antibody is capable ofbinding an epitope in a defined sequence region of a specific protein,the at least one antibody not being capable of specifically bindingepitopes of the protein outside of the defined region.

According to the invention, an antibody which is capable of binding anepitope may be used to determine proBNP or fragments thereof.Nevertheless, it may be advantageous to use several (e.g. two, three,four or five) antibodies which are capable of binding different epitopesof the proBNP.

The determination of the presence, or concentration, respectively, ofthe feline or canine proBNP, or fragment thereof, present in the samplecan be effected by methods known in the prior art. By way of example,the carrying out of enzyme immunoassays (e.g. ELISA) may be mentioned incase of liquid samples or immunohistochemical methods in case of tissuesamples.

“Antibodies” according to the present invention also comprise fragmentsof antibodies which are capable of recognizing an epitope according tothe invention. Thus, an antibody may, e.g., merely consist of the F(ab)portion which exhibits the antigen-binding side. These antibodyfragments may furthermore be part of a bispecific antibody or of aheterominibody (cf., e.g., EP 1 100 830 B1).

“proBNP or their fragments” according to the invention comprise all theproBNP fragments which are formed in vivo (e.g. Nt-proBNP) or in vitro(e.g. by mixing a sample with protease or with chemical substances, suchas CNBr), and which have the epitopes according to the invention.

According to a preferred embodiment, the at least one antibody binds toat least one epitope in the region comprising the amino acids 25-35and/or in the region comprising the amino acids 45-55 and/or in theregion comprising the amino acids 60-80 of the feline proBNP.

It has been shown that primarily the above-indicated amino acid regionsof the feline proBNP have epitopes which allow for a specific binding ofantibodies.

In a method according to the present invention, several antibodies canbe used which are capable of specifically binding several differentepitopes on feline, or canine, respectively, proBNP. For this reason, atleast one antibody which is capable of binding to at least one epitopecan be used according to the invention. Furthermore, it should bementioned that the amino acid regions indicated here may comprise notonly one epitope, but, depending on their size, may comprise severalepitopes. Thus, the method according to the invention comprises the useof a combination of several antibodies which are capable of specificallybinding to at least one epitope.

According to the invention, when determining canine proBNP or fragmentsthereof, the at least one antibody binds to at least one epitope in theregion comprising the amino acids 25-41 and/or in the region comprisingthe amino acids 55-65 and/or in the region comprising the amino acids74-86 of the canine proBNP.

Antibodies which recognize epitopes in these regions are particularlywell suited for determining proBNP or the fragments thereof in a sampleof canine origin.

According to a preferred embodiment, the at least one epitope comprisesat least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, at least ten amino acids.

According to a preferred embodiment, the at least one antibody ispolyclonal and/or monoclonal.

The antibodies employed in a method according to the invention may bepolyclonal as well as monoclonal. For preparing these antibodies,peptide fragments comprising the amino acid regions disclosed herein ofthe feline and/or of the canine proBNP are used. These peptide fragmentsmay be produced either synthetically, (Merrifield R. P., 1963, J Am ChemSoc 85, 2000, 149), recombinantly, or by chemical or enzymaticdegradation of proBNP of recombinant or native origin. Depending ontheir size, the peptides recovered therefrom will be bound to animmunogenic carrier (e.g. KLH) or directly be used for preparingpolyclonal or monoclonal antibodies (e.g. Köhler G. and Milstein C.,1975, Nature 256:495; Galfre et al., 1977, Nature 266:550). According tothe invention, the antibodies may also be recombinantly prepared. Methodfor preparing recombinant antibodies are sufficiently known to theperson skilled in the art (cf., e.g., Sambrook et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor, Laboratory Press,2001).

According to a further preferred embodiment, at least one furtherantibody binds to the at least one antibody or to the at least oneepitope, whereby, for instance, it is rendered possible to carry out theinventive test as a sandwich assay.

The binding of a further antibody to the at least one antibody makes itpossible to determine the latter and indirectly, the epitope bound tothe at least one antibody qualitatively and quantitatively,respectively. If the at least one further antibody binds to the at leastone epitope, it is possible to determine the binding of the at least oneantibody to the at least one epitope qualitatively and quantitatively,respectively, via an enzyme immuno assay, e.g. if the at least oneantibody is immobilized on a solid phase.

Preferably, the at least one antibody and/or the at least one furtherantibody is labelled.

In doing so, the at least one antibody and/or the at least one furtherantibody is labelled with an enzyme, such as peroxidase, in particularhorseradish peroxidase, biotin, fluorecent dye, in particularfluorescein (FITC, DFTF), R-phycoerythrin (PE), peridinium chlorophyllprotein (PerCP) and tandem conjugates, such as PE-Cy5 or PE-Texas Red,gold colloid or radionuclides.

By labelling one of the two antibodies, it is possible to determine in asecondary reaction, or also directly, the presence and/or theconcentration of the labelled antibody bound to the at least oneepitope. The antibodies themselves again can be detected by protein Aconjugates (e.g., protein A gold conjugate).

According to a preferred embodiment, the at least one antibody or the atleast one further antibody is bound to a solid phase.

By the binding of the at least one antibody or of the at least onefurther antibody, it is possible to produce, e.g., antibody chips,coated microtiter plates or lateral flow devices which can be used in agreat number of methods.

Preferably, the determination of feline or canine proBNP or of fragmentsthereof is effected by a method selected from the group consisting ofradioimmunoassay, immune binding assay, Western blot,immunohistochemistry, enzyme immunoassay, lateral flow device (LFD, teststrips), and combinations thereof.

The above-mentioned methods are sufficiently known to the person skilledin the art. A survey of these methods is given e.g. in “Bioanalytik”(Lottspeich and Zorbas, Spektrum Verlag 1998). Lateral flow devices(LFD, test strips) are disclosed, e.g., in WO 02/059567.

According to a further aspect, the present invention relates toantibodies or antibody mixtures binding to at least one epitope in theregion comprising the amino acids 20-42 and/or in the region comprisingthe amino acids 57-80 of feline proBNP.

According to a further preferred embodiment, the antibodies or theantibody mixtures bind to at least one epitope in the region comprisingthe amino acids 25-35 and/or in the region comprising the amino acids45-55 and/or in the region comprising the amino acids 60-80 of thefeline proBNP.

According to a further aspect, the present invention relates to anantibody or to an anti-body mixture which binds to at least one epitopein the region comprising the amino acids 20-86 of the canine proBNP.

Preferably, the antibody or the antibody mixture binds to at least oneepitope in the region comprising the amino acids 25-41 and/or in theregion comprising the amino acids 55-65 and/or in the region comprisingthe amino acids 74-86 of the canine proBNP.

According to a preferred embodiment, the antibody or the antibodymixture binds to an epitope which comprises at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, at least ten amino acids. The epitopes according to theinvention preferably have a length of 40 amino acids at the most, 35amino acids at the most, 30 amino acids at the most, in particular 25amino acids at the most, 20 amino acids at the most, or 15 amino acidsat the most.

A further aspect of the present invention relates to a peptidecomprising three amino acids in the region of the amino acids 20-42and/or in the region comprising the amino acids 57-80 of the felineproBNP.

According to a further embodiment, the peptide comprises at least threeamino acids in the region of the amino acids 25-35 and/or in the regionof the amino acids 45-55 and/or in the region of the amino acids 60-80of the feline proBNP.

A further aspect of the present invention relates to a peptidecomprising three amino acids in the region of the amino acids 20-86 ofthe canine proBNP.

Preferably, the peptide comprises at least three amino acids in theregion of the amino acids 25-41 and/or in the region comprising theamino acids 55-65 and/or in the region of the amino acids 74-86 of thecanine proBNP.

According to a preferred embodiment, the peptide is chemicallysynthesized or isolated from a sample, or prepared recombinantly,respectively.

In order to appropriately prepare the epitope from a peptide which hasbeen isolated from a sample or has been recombinantly produced,respectively, the latter may be further processed by enzymatic orchemical methods known per se.

A further aspect of the present invention relates to the use of aninventive antibody or of an antibody mixture for determining feline orcanine proBNP or fragments thereof in the method according to theinvention.

The peptides according to the invention can be used in competitiveimmunoassays in labelled form.

Preferably, the peptides according to the present invention are used forpreparing an anti-body or an antibody mixture.

Furthermore, the peptides according to the present invention are used aspositive control or as a standard, respectively, for concentrationdeterminations in a method according to the invention.

A further aspect of the present invention relates to a kit fordetermining feline or canine proBNP or fragments thereof, comprising atleast one antibody according to the invention or at least one antibodymixture according to the invention, means for the qualitative and/orquantitative detection of a binding of the at least one antibody or ofthe at least one antibody mixture to feline or canine proBNP orfragments thereof, and, optionally, peptides according to the inventionand/or feline or canine proBNP or fragments thereof as a positivecontrol or as a standard for a concentration determination.

According to the invention, the kit may comprise at least one furtherantibody.

This additional antibody has an avidity to at least one antibody, oralso to the at least one epitope.

According to a preferred embodiment, the at least one antibody and/orthe at least one further antibody is labelled.

Preferably, the labelling comprises enzymes, such as peroxidases, inparticular horseradish peroxidase, biotin, fluorecent dye, in particularfluorescein (FITC, DFTF), R-phycoerythrin (PE), peridinium chlorophyllprotein (PerCP) and tandem conjugates, such as PE-Cy5 or PE-Texas Red,gold colloid or radionuclides.

A further aspect of the present invention relates to the use of a kitaccording to the invention in a method of determining feline or canineproBNP.

With the method of the present invention, it is possible not only todetect proBNP and its fragments in cats and dogs, but also in othermammals, such as horses, cattle, elephants, mice (Swiss-Prot: P40753),pigs (Swiss-Prot: P07634), rats (Swiss-Prot: P13205), camels(Swiss-Prot: Q6L7Z3) and sheep (Swiss-Prot: 046541) and fish, such asperch (Swiss-Prot: Q805E8), sturgeon (Swiss-Prot: P83965) and pufferfish(Swiss-Prot: Q805D7).

In order to detect proBNP in the above-mentioned animals, antibodieswhich bind to at least one epitope in an amino acid region of amino acidresidue 1 to amino acid residue 80 of the corresponding proBNP arepreferred. In particular antibodies are preferred which bind to at leastone epitope comprising the amino acid regions 1-15, 15-30, 20-30, 25-35,30-40, 35-50, 35-55, 45-55, 50-70, 60-70, 60-80 and 70-80. The followingparticularly preferred specific epitopes have also been found with thescheme lying at the basis of the present invention (cf. Examples).

TABLE 1 Swiss-Prot No. AA-Region Mouse P40753 1-15, 20-30, 50-70 PigP07634 20-30, 35-50, 60-70 Rat P13205 1-15, 30-40, 45-55, 60-80 CamelQ6L7Z3 20-30, 55-65, 70-80 Sheep O46541 1-15, 20-30, 45-55, 60-80 PerchQ805E8 15-30, 35-50, 70-80 Sturgeon P83965 1-20, 25-35, 70-80 PufferfishQ805D7 15-30, 35-55, 60-80

As listed above for dogs and cats, the preferred lengths of the epitopesare also given for the above-mentioned animals.

The present invention is further illustrated by the following Examplesand Figures without, however, being restricted thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows epitope recognition factors corresponding to the programProtScale of the canine proBNP amino acid sequence (FIG. 1A) and aminoacid sequences of the inventive canine proBNP epitope (FIG. 1B) (SEQ IDNOs: 1-5).

FIG. 2 shows epitope recognition factors of the feline proBNP amino acidsequence, calculated with ProtScale (FIG. 2A) and amino acid sequencesof the inventive feline proBNP epitopes (FIG. 2B) (SEQ ID NOs: 6-9).

FIG. 3 shows standard curves based on ELISA assays using inventiveantibodies with canine (FIG. 3A) and feline (FIG. 3B) proBNP. It couldimpressively be demonstrated that the proBNP determination with theinventive antibodies is linear over a wide concentration range.

FIG. 4 shows the determination of proBNP in 47 sick and 28 healthy cats.The concentration of proBNP in the samples allows for determining theseverity of the disease. FAT—feline atriale thrombosis,HCMP—hypertrophic cardiomyopathy, LVH—left ventricular hypertrophy.

DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS Examples Example 1Preparing the Antibodies

Epitopes may, e.g. be determined by calculation with ProtScale(http://www.expasy.org/tools/protscale.html) according to the algorithmof Fraga S. (“Theoretical prediction of protein antigenic determinantsfrom amino acid sequences.”, 1982; Can. J. Chem. 60:2606-2610).

The peptide fragments were chosen from those regions of the amino acidsequence of the feline or canine Nt-proBNP, in which a maximum of theepitope recognition factors (corresponding to the results of theProtScale program) was obtained, since these epitopes proved to beparticularly immunogenic and readily accessible for antibodies. Theselected peptides of the feline or canine proBNP were chemicallysynthesised and conjugated to a suitable carrier protein (KLH, e.g.).

One peptide/epitope each, conjugated to KLH, was injected into threesheep. For the first immunisation, each sheep received 0.5 mg of thecorresponding antigen, mixed with Freund's Adjuvant (Guildhay, UK) andBCG (Bacillus Calmette-Guérin) and 0.25 mg of the immunogens to furtherincrease the immune response.

According to the invention, it has been shown that the use of polyclonalantibodies yields very good and reproducible results. Nevertheless, theuse of monoclonal antibodies in a method such as described in thepresent invention is also possible. Monoclonal antibodies againstpeptides/epitopes of the feline or canine proBNP can be prepared bystandard methods known to the person skilled in the art (cf. in thisrespect e.g. Köhler G and Milstein C, Nature, 1975, 256:495-497).

Example 2 Determining the Antibody Reactivity by Means of ELISA

The reactivity of antibodies or sera, respectively, againstpeptides/epitopes of the proBNP which are recovered from the blood ofthe sheep was assayed by means of an ELISA test. At first, themicrotiter plates were coated over night at 4° C. with Streptavidin (0.5μg/ml, 200 μl per well), washed, blocked with 1% BSA in 0.1 M PBS, pH7.5, containing 0.25% Tween 20, washed once more and incubated for 3 hat 4° C. with synthetic proBNP peptide sequences conjugated to biotin(0.25 μg/ml, 200 μl per well). After a further washing step, the serumsamples were diluted 1:1000/1:10000/1:100000 with 0.1 M phosphate buffercontaining 3% BSA and applied to the microtiter plate. The binding ofthe antibodies to the peptides/epitopes on the plate was determined bythe addition of anti-sheep-IgG antibodies which are conjugated withhorseradish peroxidase, and of a substrate solution comprising TMB(tetramethyl-benzidine). The reaction of the horseradish peroxidase withTMB was stopped by adding 0.9% sulfuric acid. The colour development wasmonitored with a photometer capable of analyzing microtiter plates.

Example 3 Nt-proBNP Measurement in Samples of Healthy and Sick Animals

Into the wells of a microtiter plate coated with one of the inventiveantibodies, 20 μl of feline or canine serum were pipetted and incubatedfor 4-16 h at room temperature with 200 μl of a second, furtherperoxidase-labelled inventive antibody in 0.1 M phosphate buffer, pH 7.Subsequently, the microtiter plate was washed with 5×300 μl of 0.1 Mphosphate buffer, pH 7, with 0.1% triton×100, and 200 μl of tetramethylbenzidine were added as substrate. After a colour development of 20-30minutes, the reaction is stopped by adding 50 μl of 0.9% sulfuric acid,and the colour intensity which is directly proportional to the amount ofNt-proBNP is measured with a microtiter plate photometer. The exactconcentration is determined by a comparison with a calibration curvefrom recombinant feline or canine Nt-proBNP.

By way of example, in 8 healthy dogs and in 15 dogs suffering from heartdisease the concentration of Nt-pro-BNP was determined by means ofantibodies against the epitopes in the region of the amino acids 25-41and 74-86 of the canine Nt-proBNP (Table 2):

TABLE 2 Canine Nt-proBNP State of Health No. pmol/l healthy 1 862 2 10603 753 4 531 5 980 6 674 7 695 8 1010 suffering from heart disease 432460 44 1950 45 2140 46 2170 47 1480 48 1560 49 1390 50 1450 51 1520 521790 53 1310 54 1140 55 1975 57 1720 58 3020

These results show that with the antibodies of the present invention,the concentration of Nt-proBNP can efficiently be determined in theserum of animals, and with the results therefrom, a diagnosis on thestate of health can be established, or the course of a therapy can bemonitored, respectively.

Furthermore, FIG. 4 shows the determination of Nt-proBNP in 47 sick and28 healthy cats. It was found that the concentration of detectedNt-proBNP directly correlates with the severity of the heart disease. Inthese tests, antibodies, which bind epitopes in the region comprisingthe amino acids 35 to 45 and 68 to 80 were used. The results obtainedconfirm numerous publications in which a similar connection had beenpostulated.

Example 4 Cross Reactivity

Recombinant feline, canine and human Nt-proBNP were coated on microtiterplates (250 ng/ml, 200 μl/well, over night, room temperature).Subsequently, the plates were washed and contacted with a dilution ofthe anti-human, anti-feline and anti-canine antisera (10-100 μg/ml, in0.1 M phosphate buffer, pH 7). After a washing step, the amount of boundantibodies was measured with a suitable secondary antibody(peroxidase-labelled anti-sheep antibody). It has been shown that therespective antibodies react very well with the corresponding Nt-proBNPmolecules (i.e., anti-feline antiserum with feline Nt-proBNP), butsurprisingly do not react or react to a very slight extent with theNt-proBNP molecules of the respective other species.

It could be demonstrated that the antibodies which were produced againstthe epitopes of the feline Nt-proBNP exhibit a high specificity and canbind to the corresponding human sequence to a very slight extent only.Since in the measurement of the antibody specificity Nt-proBNP was usedas entire polypeptide as the binding partner and not the peptides whichwere used for producing the antibodies, it could impressively bedemonstrated that the antibodies to feline epitopes of the Nt-proBNP donot exhibit any cross-reaction over the entire sequence region of thehuman Nt-proBNP. An exception is only that antibody which binds in theregion of the amino acids 1 to 20 of feline Nt-proBNP. When binding tofeline Nt-proBNP, this antibody shows only twice as high a relativereactivity than when binding to human Nt-proBNP.

Moreover, it could be demonstrated that antibodies against humanepitopes of the Nt-proBNP also have a low reactivity relative to felineNt-proBNP. Thus, it could be impressively be demonstrated thatantibodies which are directed against epitopes of the human Nt-proBNP,cannot bind to feline Nt-proBNP and, thus, cannot be employed fordetermining Nt-proBNP in cats (cf. Table 3).

TABLE 3 Rel. Reactivity Relative Antiserum Relative to the CorrespondingNo. Antibody Specificity Reactivity Human Sequences S2189 AA 1-20 feline2.3 1.2 S2190 AA 45-55 feline 3.7 0.01 S2191 AA 25-35 feline 1.0 0.2S2192 AA 60-80 feline 4.2 0.3 S2072 AA 8-29 human 0.4 — S2104 AA 32-57human 0.25 — S2102 AA 60-80 human 1.7 —

The cross-reactivity was also tested with antibodies against epitopes ofthe canine Nt-proBNP and with antibodies against epitopes of the humanNt-proBNP. Also with canine sequences, a result comparable to the testswith feline sequences could be achieved (cf. Table 4)

TABLE 4 Rel. Reactivity Relative Antiserum Relative to the CorrespondingNo. Antibody Specificity Reactivity Human Sequences S2195 AA 1-22 canine2.2 1.1 S2196 AA 25-41 canine 6.3 0.2 S2197 AA 55-65 canine 1.0 0.03S2198 AA 74-86 canine 1.9 0.6 S2072 AA 8-29 human 0.1 — S2104 AA 32-57human 0.3 — S2102 AA 60-80 human 1.5 —

1. A method of detecting canine proBNP or a fragment thereof in a caninesample comprising (a) providing a canine sample; (b) contacting thesample with at least one antibody that binds an epitope in the regionfrom amino acids 1 to 22 of canine proBNP; and (c) detecting the bindingof the antibody to the epitope whereby the canine proBNP or the fragmentthereof in the sample is detected.
 2. The method of claim 1, wherein theepitope comprises at least 3 amino acids.
 3. The method of claim 1,wherein the at least one antibody is polyclonal.
 4. The method of claim1, wherein the at least one antibody is monoclonal.
 5. The method ofclaim 1, wherein the sample is a blood sample or a urine sample.
 6. Themethod of claim 1, wherein detecting the binding of the antibody to theepitope comprises radioimmunoassay, immune binding assay, Western blot,immunohistochemistry, or enzyme immunoassay.
 7. The method of claim 1,further comprising determining the concentration of the canine proBNP orfragment thereof in the sample.
 8. The method of claim 7, furthercomprising comparing the concentration of the canine proBNP or fragmentthereof in the sample with the concentration of canine proBNP orfragment thereof in a sample from a healthy canine.
 9. The method ofclaim 8, further comprising diagnosing cardiac insufficiency in a caninefrom which the sample was obtained if the concentration of the canineproBNP or fragment thereof in the sample is higher than theconcentration of canine proBNP or fragment thereof in the sample fromthe healthy canine.
 10. The method of claim 1, wherein contacting thesample with the at least one antibody occurs on a solid phase.
 11. Themethod of claim 10, wherein the at least one antibody is bound to asolid phase.
 12. The method of claim 10, wherein the canine proBNP orfragment thereof is bound to a solid phase.
 13. An isolated antibodythat binds an epitope in the region from amino acids 1 to 22 of canineproBNP.
 14. The antibody of claim 13, wherein the epitope comprises atleast 3 amino acids in the region from amino acids 1 to 22 of canineproBNP.
 15. A kit for detecting canine proBNP or a fragment thereofcomprising an antibody that binds an epitope in the region from aminoacids 1 to 22 of canine proBNP and a label.
 16. The kit of claim 15,wherein the label is peroxidase, biotin, fluorescent dye, gold colloid,or a radionuclide.
 17. The kit of claim 15, further comprising a peptidecomprising amino acids 1 to 22 of canine proBNP.
 18. The kit of claim15, further comprising at least one further antibody.
 19. The kit ofclaim 18, wherein the at least one further antibody is labeled.
 20. Thekit of claim 15, wherein the antibody is bound to a solid phase.